Cardiovascular disease (CVD) is the number one cause of death in the United States and ischemic heart disease is the leader in mortality among patients with CVD. Most individuals with ischemic heart disease have blocked coronary arteries and cardiac tissue has a lack of perfusion (ischemia), resulting in myocardial damage and further injury following reperfusion. The novel CYP-epoxygenase metabolites of arachidonic acid (AA), 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) are increased during ischemia and particularly following reperfusion. Endogenously produced EETs and exogenously administered EETs produce marked cardioprotective effects in dog, rat and mouse hearts; however, the mechanisms responsible for these beneficial effects remain unclear. Based on intriguing preliminary data, opioid receptors and nitric oxide (NO) release may be 2 major players in EET-mediated cardioprotection. This study will utilize rats and genetic knockout mice of nitric oxide synthase (NOS) isoforms as well as cellular models of cardiomyocytes and cardiac fibroblasts to elucidate in depth, the key contributing factors responsible for EET-induced cardioprotection. The hypothesis to be tested is that upon ischemia/reperfusion, EETs released from cardiomyocytes and cardiac fibroblasts are potent cardioprotective agents that induce the further release of endogenous NO to produce their beneficial effects. Specifically, we will (1) determine that nitric oxide (NO) is a mediator of EET-induced cardioprotection in intact rat and mouse hearts and cardiomyocytes. Both rats and NOS knockout mice and cardiomyocytes (H9c2 cells) will be used to demonstrate that EET-induced cardioprotection is mediated via a NO signaling pathway. NOS isoforms (eNOS, nNOS or iNOS) and their signaling pathways activated by the EETs will be identified. (2) Determine that cardiac fibroblasts play a role in releasing EETs and possibly NO as regulatory factor(s) to protect cardiomyocytes from hypoxia/reoxygenation injury. (3) Determine if cross-talk occurs between the two major cardioprotective factors, EETs and opioids, in cardioprotection. Importantly, EETs are readily hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which have no effect on myocardial infarct size. This finding suggests that novel synthetic EET analogs with superior pharmacokinetics may represent better therapeutic targets than sEH inhibitors and labile EETs. The newly synthesized longer-acting EET analogs will be used to elucidate signaling pathways of the EETs and to reduce infarct size. As leaders in the this field, identification of novel stable analogs of endogenous EETs will be a major goal of this project as well as demonstrating their powerful anti-ischemic and anti-inflammatory actions in cellular and whole animal models of cardiac injury. The long term goal is to obtain better characterization of a novel endogenous system with multiple therapeutic targets that may suggest combined therapy for better treatment of ischemia/reperfusion injury.